Otoliths of Caspian gobies (Teleostei: Gobiidae): Morphological diversity and phylogenetic implications

Otoliths (ear stones) of the inner ears of teleost fishes, which develop independently from the skeleton and are functionally associated with hearing and the sense of equilibrium, have significantly contributed to contemporary understanding of teleost fish systematics and evolutionary diversity. The sagittal otolith is of particular interest, since it often possesses distinctive morphological features that differ significantly among species, and have been shown to be species- and genus-specific, making it an informative taxonomic tool for ichthyologists. The otolith morphology of the Caspian Sea gobiids has not been thoroughly studied yet, with data available for only a few species. The aim of the present paper is to examine the qualitative and quantitative taxonomic and phylogenetic information in the sagittal otoliths of these species. A total of 118 otoliths representing 30 gobiid species (including 53.5% of the Caspian gobiofauna) in three gobiid lineages (i.e., Gobius, Pomatoschistus, and Acanthogobius) and 11 genera (i.e., all Ponto-Caspian gobiid genera except Babka) were analysed at taxonomic levels using an integrated descriptive and morphometric approach. The results indicated high taxonomic efficiency of otolith morphology and morphometry at taxonomic levels for the Ponto-Caspian gobiids. Our qualitative and quantitative otolith data also (i) support the monophyly of neogobiin gobies, (ii) along with other morphological and ecological data, offer a new perspective on the systematics of Neogobius bathybius, (iii) suggest the reassignment of Hyrcanogobius bergi to the genus Knipowitschia, and (iv) question the phylogenetic integrity of the four phenotypic groups previously defined in the tadpole-goby genus Benthophilus; however, more studies are needed to complete these evaluations and confirm our otolith study findings.


Introduction
The otoliths of the inner ears in teleost fishes are arranged in three pairs termed the saccular (sagittae, the largest otoliths in most teleosts), utricular (lapilli), and lagenar (asterisci) otoliths.

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Otolith data analysis
The otoliths were analysed based on two different approaches: (i). A descriptive method for all 30 species.
(ii). A quantitative method based on otolith morphometric variables and shape descriptors for which comparisons were made at four levels: lineage, tribe, genus, and species (Table 1). In the genus-and species-level analyses, genera and species with less than five otoliths were not considered. All otolith variables including morphometric ratios, inclination angles, and shape indices were analysed using IBM SPSS Statistics 26.0 [48]. The normal distributions of otolith variables for each species, genus, tribe, and lineage were compared using Shapiro-Wilk tests (p > 0.05). Mann-Whitney tests (p < 0.05) were used to evaluate the significance of non-normally distributed otolith variables; Univariate Analysis of Variance (ANOVA) with Tukey's HSD (p < 0.05) and Dunnett's T3 (p < 0.05) post-hoc tests (depending on homogeneity of variances, Levene's test, Knipowitschia longecaudata (Kessler, 1877) p > 0.05) were used for taxon comparisons of normally distributed otolith variables. A Bonferroni correction (0.05/number of tests) was used at each taxonomic level to correct for multiple tests. Discriminant function analysis (DFA) was conducted to determine the proportion of otoliths that could be correctly assigned to their corresponding species, genera, tribes, and lineages. The classification success was tested by leave-one-out cross validation. A dendrogram was constructed based on Euclidean distance as a measure of dissimilarity to show the phenotypic relationships among the species. The between groups linkage method was used as the clustering algorithm.

Molecular phylogeny
There have been a few efforts to assess molecular phylogenetic relationships among benthophilines [29,30,61]. Neilson & Stepien [29] were the first to estimate relationships within this group; they used a combined dataset of two mitochondrial (cyt b and COI) and two nuclear loci (RAG1 and S7) with maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) approaches. They introduced a revised classification and nomenclature that does not, however, aptly fit with the present understanding of gobioid family and subfamily phylogenetics (e.g., presented in Agorreta et al. [28] and McCraney et al. [60]), however, their primary phylogenetic inferences and tribe designations remain helpful as they are not in disagreement with any familial and subfamilial systematics. However, the most comprehensive phylogenetic analysis of the benthophiline gobies in terms of specimens and species numbers and also geographic coverage was conducted by Zarei et al. [30] based on mitochondrial COI sequences using ML and BI approaches, on which the current classification was based. That mitochondrial phylogeny is similar to the combined mito-nuclear phylogeny of Neilson and Stepien [29], except for (i) slightly different placement of Babka; and (ii) with regard to the sister group relationship of Ponticolini with Neogobiini rather than with Benthophilini; that grouping was, however, not robustly supported in Neilson and Stepien [29].
Benthophilus durrelli. Long elliptical (Fig 2I, from Schwarzhans et al. [45]); OL/OH 1.4; dorsal rim gently curved, mostly entire with a shallow incision in mid-length, highest behind incision above cauda, anteriorly slightly depressed; predorsal angle well-developed, broad and round; posterodorsal projection broad and long, rounded at tip; anterior rim little oblique, β 82.9˚, incised slightly above the level of ostium; posterior rim more oblique comparing to the anterior rim, γ 108.1˚, with a broad incision at the level of cauda; δ 19.1; ventral rim nearly horizontal or gently curving, entire; preventral projection short and broad, rounded at tip; posteroventral angle slightly projected, entire; sulcus centrally positioned, sole-shaped, horizontal, shallow, ostial lobes weakly developed; OL2/CL 2.4; subcaudal iugum absent; ventral furrow runs with a moderate distance to ventral rim; dorsal depression distinct and relatively narrow.
Mesogobius nunultimus. Two-humped long rectangle ( Fig 4D); OL/OH 1.3; dorsal rim two-humped with a deep broad V-shaped concavity at mid-length (i.e., M-shaped dorsal rim), humps angular, posterior hump markedly broader and higher, outline entire to sinuate; posterodorsal projection bulky and very broad, not bending outwards; anterior rim oblique, β 73.7˚, with a small incision above the level of ostial apex, entire; posterior rim almost parallel to the anterior rim or slightly less oblique, γ 101.6˚, with a shallow small incision below the level of cauda; δ 27.2˚; ventral rim almost horizontal, entire; preventral projection very long and pointed; posteroventral angle rounded, entire; sulcus centrally positioned, sole-shaped, α 17.5˚, relatively deep, ostial lobes developed; OL2/CL 1.7; subcaudal iugum present, its length 1/2 cauda length and slender; ventral furrow runs with a moderate to large distance to ventral rim; dorsal depression indistinct.

Otolith morphometric variables and classical shape descriptors
Variation among the lineages. Twenty-eight otolith variables significantly differed between two or more of the three lineages (Table 2, S1 Fig, S1 Table). Fourteen variables were
Variation among the genera. Thirty-one otolith variables revealed significant differences between at least two of the seven genera (Table 6, S3 Fig, S3 Table). Thirteen variables were normally distributed: four variables involved the SuL, four the SuH, and the remainder were γ, OL/OH, OP/OH, SuEndV/OP, and SuH/SuP. Eighteen variables were non-normally distributed: three inclination angles (α, δ, and β), three involved the SuL, three the SuP, two the SuH, two shape indices (ROx and ELx), and the remainder were OP/OL, SuA/OA, SuTipV/OP, SuTipV/SuEndV, and OL2/CL.
Variation in the Pomatoschistus and Acanthogobius lineages. Twenty-seven otolith variables differed between at least two of the four studied species from the Pomatoschistus and Acanthogobius lineages (Table 11, S6 Fig, S4 Table). Twenty variables were normally distributed: five variables involved the SuL, four the SuH, two the SuP, three inclination angles (α, γ, and δ), and the remainder were SuTipV/OP, REx, OP/OH, SuA/OA, OL2/CL, and SuEndV/ OP. Seven variables were non-normally distributed: two involved the SuL, and the remainder were SuP/OP, SuH/SuTipV, SuTipV/SuEndV, OL/OH, and ELx.

PLOS ONE
Otoliths of Caspian gobies (Teleostei: Gobiidae) Phenotypic relationships based on otolith variables. A linkage dendrogram based on average Euclidean distances was calculated for 31 otolith variables (Fig 9), which separated the studied fish species into three groups. Group I contained the species in the Pomatoschistus and Acanthogobius lineages as well as Proterorhinus nasalis of the Gobius lineage. Group I itself included two sub-groups, with one containing species in the Pomatoschistus lineage and

PLOS ONE
Otoliths of Caspian gobies (Teleostei: Gobiidae) Acanthogobius lineage, and the second with Proterorhinus nasalis. Within the first sub-group, species of the Pomatoschistus lineage were monophyletic, sister to Rhinogobius sp. The dendrogram shows a closer phenotypic relationship of Knipowitschia longecaudata with Hyrcanogobius bergi, rather than with K. caucasica. Group II included all benthophiline gobies except for Proterorhinus nasalis. Four subgroups were resolved within Group II. The basal sub-group was Benthophilus leobergius and B. pinchuki. Species in the genus Neogobius were arranged in two sub-groups: one with N. pallasi, N. caspius, and N. melanostomus, and the other of N. bathybius alone. The fourth sub-group included all species in the genus Ponticola with two sub-clusters: (i) the Ponticola syrman group comprising P. syrman, and two freshwater endemic species, P. iranicus and P. patimari, and (ii) the Ponticola kessleri group comprising P. iljini, P. kessleri and P. gorlap. Neogobius bathybius occupied an intermediate position between the Neogobius and Ponticola sub-groups.

Taxonomic significance of otolith morphology in the Caspian gobies
This study aimed to compare the otoliths of extant Caspian gobiids at different taxonomic levels based on detailed descriptions and morphometric method to reveal the taxonomic and phylogenetic information inscribed in their otoliths. The results showed that the otoliths belonging to the Gobius lineage are significantly different from those of the Pomatoschistus and Acanthogobius lineages based on general shape, characteristics of anterior and posterior rims, and otolith variables (overall, 22 otolith variables distinguished the Gobius lineage from the other two lineages). Based on morphometric otolith variables, otoliths of the Pomatoschistus and Acanthogobius lineages from the Caspian Sea basin were largely indistinguishable from each other, separated by small differences in inclination of line connecting preventral angle with tip of posterodorsal projection and inclination angle of posterior rim (see Table 2, S1 Fig, S1 Table). Otolith descriptions however, showed that the otoliths of the two lineages also differed in the shape of their dorsal rim, i.e., being more angular in the Pomatoschistus lineage. These results appear to be consistent with the phylogenetic relationships among the three lineages [28]. The overall rate of classification success at this taxonomic level was 85.4%, but 91.3% for the Gobius lineage. Within the Gobius lineage, the otoliths of the three benthophiline tribes were clearly different based on both methods, with an overall classification success of 94.2%.
The results indicated that the overall shape of the otolith is genus-specific for all Ponto-Caspian gobiid genera, except for Hyrcanogobius and Knipowitschia, making it the most efficient otolith characteristic for gobiid genus identification in the Caspian Sea basin. The monotypic genus Hyrcanogobius and Knipowitschia were separated by slight differences in just four variables, i.e., SuL/SuP, α, δ, and REx. Hyrcanogobius bergi was originally described by Iljin [50] from the river mouths of the north Caspian Sea as a separate genus Hyrcanogobius, because of the reduced condition of the head lateral-line canal system, but later suggested to be congeneric with Knipowitschia by Economidis and Miller [51]. However, according to Miller [52], the possession of transverse interorbital papillae rows and a much greater extent of anterior transverse oculoscapular row tra (extending downwards to or near the longitudinal suborbital row b vs. noticeably short of row b in Knipowitschia) appear to warrant recognition as a separate genus in any classification based on the head lateral-line system. In addition, paleontological data (otolith) by Bratishko et al. [53] suggest that Hyrcanogobius is recognizable as a separate lineage in the fossil record since 11 million years ago. Molecular data of H. bergi are still missing, however, otolith data support the reassignment of H. bergi to the genus Knipowitschia Iljin, 1927, hereby highlighting the necessity of an integrative molecular, morphological, and paleontological analysis on these species to evaluate their taxonomy.
Neogobius presently comprises five valid species: N. caspius, N. pallasi, and N. bathybius are Caspian endemics, N. fluviatilis in the Black Sea is a sister species of the Caspian N. pallasi, and N. melanostomus is native to the Ponto-Caspian basins. The four Caspian species which were well-represented in this study, can be easily distinguished from one other based on otolith morphology results from both descriptive and morphometric methods. The otolith of N. fluviatilis however, which is poorly represented in this study, is very similar to those of the Caspian N. pallasi, but a preliminary description of the N. fluviatilis otolith suggests that they differ with regard to the shape of posterodorsal projection (long and tapering in N. fluviatilis vs. short and rounded in N. pallasi) and sulcus (α 14.3˚in N. fluviatilis vs. 16.0-22.5˚in N. pallasi; shallow and ostial lobes weakly developed in N. fluviatilis vs. relatively deep and ostial lobes well-developed in N. pallasi). Obviously, more data on the otolith of N. fluviatilis is needed before drawing any conclusion about its otolith morphology. Gobius fluviatilis Pallas, 1814 was originally described in part from near the mouths of rivers falling into the Black Sea and similarly the Caspian Sea. Neogobius fluviatilis pallasi (Berg, 1916) was the subspecies described in the Caspian Sea basin. Kottelat and Freyhof [54] recognized N. pallasi as the Caspian Sea species and restricted N. fluviatilis to the Black Sea basin. This taxonomic decision was later confirmed by molecular data [55].
Ponticola presently comprises eight recognized species in the Caspian Sea basin, six of which were included in this study. The overall shape of otolith in these species is invariably long parallelogram, however, the otoliths of P. syrman and P. hircaniaensis are easily distinguishable. The otoliths of P. syrman and P. hircaniaensis show concavity in their dorsal rim, however, they are different in their OL/OH (1.  [5]. The otoliths of P. gorlap differ from those of two south Caspian freshwater endemic species, P. iranicus and P. patimari in five (γ, SuP/SuTipV, SuL/SuTipV, REx, and SuA/OA) and four (SuP/SuTipV, SuL/SuTipV, SuTipV/OP, and OL2/CL) otolith variables, respectively. The otoliths of P. gorlap also differ from those of both species with regard to the incision below the posterodorsal projection (markedly incised vs. slightly incised or not incised), the shape of the predorsal angle (broadly rounded vs. obtuse or orthogonal) and dorsal rim (anteriorly slightly depressed vs. anteriorly not depressed). The otoliths of P. gorlap are most similar to those of P. iljini, which may reflect their close phylogenetic relationships and morphological similarities. Ponticola gorlap was first identified in the Caspian Sea as Gobius kessleri Günther by Kessler [56], who found some morphological differences between the Caspian and typical Black Sea forms. Based on several morphological differences, Iljin [57] suggested that the Caspian gobies from the Mangyshlak region (western Kazakhstan) should be erected as a distinct species, which he described as Gobius gorlap. Later, karyological, cranial, head scale, and morphometric data of samples from the Dnieper, Dniester and Volga rivers provided the data supporting specific status of the Mangyshlak samples [58], and subsequently, it was described as a separate species by Vasil'eva and Vasil'ev [59] who regarded the species name gorlap as invalid and proposed the new name iljini, which later was synonymized with P. gorlap (Iljin) in a modern phylogenetic systematic study [29]. Vasil'eva et al. [38] reestablished the validity of P. iljini based on karyological data, but restricted its distribution to the coast of the Mangyshlak Peninsula, western Kazakhstan. As presently understood, P. kessleri, P. iljini, and P. gorlap are closely related and form independent phyletic lineages within a clade of Ponticola [29,38].
The Ponticola syrman group comprises two freshwater endemic and cryptic species distinguished from each other mainly based on molecular characters and geographic distributions [7], i.e., P. iranicus endemic to the upper Sefidroud sub-basin, and P. patimari endemic to the western freshwater habitats of the south Caspian sub-basin. PCA and DFA plots for the meristic and morphometric data also showed a clear separation of the two species [7]. Our otolith morphometric variables, inclination angles, and classical shape descriptors show that the otoliths of P. iranicus and P. patimari are only slightly different in their REx. Also, the otolith shape analysis of 213 specimens representing six sub-basin samples of these species presented a high level of shape variation which did not show congruence with their taxonomy and phylogeographic structure [7]. Studies suggest that while genetics constrain the overall shape of the otolith itself, environmental conditions may eventually alter the rates of somatic and otolith growth, which in turn may affect otolith shape.
Tadpole-gobies of the genus Benthophilus are a group of 21 poorly known species from the fresh and brackish waters of the Caspian and Black Sea basins, including the Sea of Azov [37]. Boldyrev and Bogutskaya [41] recognized 20 species and assigned them to four phenotypic groups (i.e., I, II, III, and IV), based on differences in size, arrangement and counts of dermal ossifications, fin ray counts, and body shape. However, the phylogenetic integrity of these groups has never been tested, since the phylogeny of the genus is poorly known and genetic data are available for only a few species [29,30]. The phylogenetic integrity of the four phenotypic groups established by Boldyrev & Bogutskaya [41] was questioned by Kovačić et al. [37], since a newly described species B. persicus Kovačić, Esmaeili, Zarei, Abbasi & Schliewen, 2021 featured a mix of characters of phenotypic groups II and III. Interestingly though, the phylogenetic inferences of Neilson and Stepien [29] and Zarei et al. [30] estimated a closer relationship between a group II member (a specimen identified as B. abdurahmanovi) and the group I member (a specimen identified as B. granulosus Kessler, 1877), than to three other group II species used in their analyses (identified as B. mahmudbejovi Ragimov, 1976, B. stellatus and B. leobergius). Our otolith data also question the phylogenetic integrity of the four phenotypic groups defined by Boldyrev & Bogutskaya [41]: the otoliths of the species examined here comprised four phenotypic groups based on their overall shapes, which did not show congruence to their hypothesis: group 1: B. leobergius (II); group 2: B. pinchuki (III), B. microcephalus (II), B. stellatus (II), B. baeri (IV); group 3: B. durreli (II); and group 4: B. abdurahmanovi (II). However, we consider our results preliminary and await substantially increased taxon sampling and a thorough phylogenetic analysis of the species.

Otolith data suggest the monophyly of neogobiin gobies
Molecular phylogenies have supported a monophyletic clade comprising the neogobiin and benthophilin gobies [29,60]. This monophyletic clade contains three distinctive sub-clades designated as tribes Benthophilini, Neogobiini, and Ponticolini. The phylogenetic placement of Benthophilini has been inconsistent among analyses. The two combined mito-nuclear analyses resolve Benthophilini as the sister clade to Ponticolini [29,60], however, this relationship had mixed support from different analysis methods, represented a short internal branch, having low internode certainty and gene support frequency. In the cyt b analysis of Neilson and Stepien [29], Benthophilini was the sister clade to Neogobiini, yet in their COI analysis and also the cyt b analysis of Medvedev et al. [61], Benthophilini again grouped with Ponticolini. The COI analysis of Zarei et al. [30] resolved Benthophilini as the sister clade to Ponticolini + Neogobiini (Fig 10). The latter phylogenetic hypothesis was also supported by our otolith data: Benthophilini differed significantly from Neogobiini and Ponticolini by 25 and 21 variables, respectively, whereas Neogobiini and Ponticolini were separated by 17 variables. The average linkage dendrogram based on the Euclidean distance for the otolith variables also clustered Benthophilini as the sister clade of Neogobiini + Ponticola. This phylogenetic hypothesis also agrees with Schwarzhans et al. [12] based on articulated skeletons and otoliths of fossil gobiids: (i) both the neogobiin and benthophilin subgroups were represented by ''primitive" extinct genera (i.e., †Proneogobius and †Protobenthophilus) considered to be the sister group to all extant members of their respective subgroups, and (ii) the origin and separation of the two subgroups likely links to the segregation of the Eastern Paratethys during the Langhian stage (16-13.6 Ma) of the middle Miocene. Nevertheless, additional genomic, morphological, and taxonomic sampling are needed to further resolve relationships among the major Ponto-Caspian endemic gobiid lineages.

Systematics of Neogobius bathybius
The taxonomic status of Neogobius bathybius has been controversial. Gobius bathybius Kessler (1877) originally was described from the Svinoi Island, Caspian Sea. The name Chasar appeared in print for the first time in Berg [62] as a subgenus of Neogobius to accommodate bathybius. Berg [62] provided a brief description of the species as Neogobius (Chasar) bathybius, but did not define the genus-group category. Although Vasil'eva [63] stated that Iljin [64,65] used this subgeneric name for bathybius, a search of the latter publications by Miller [66] found this species mentioned only as being incertae sedis, but without reference to any previous use of the name Chasar or to a definition by Iljin. Both Iljin [57] and Ragimov [67] used the name at a subgeneric level, but again provided no diagnosis. Pinchuk and Ragimov [68], in their redescription of bathybius, placed this species in Neogobius without a subgenus. The first subgeneric diagnosis of Chasar, with indication of the type and only species, thus appears to be by Vasil'eva [63]. Detailed osteological comparison with other gobiid taxa by Vasil'eva [63] suggested that bathybius occupied a distinct subgeneric position. The monotypic genus Chasar was recognized as a valid taxon by Miller [66], on the basis of the head sensory papillae patterns noted by Pinchuk and Ragimov [68] and Vasil'eva [63]. The resulting paraphyly of Neogobius sensu lato [62] however was changed in Neilson and Stepien's [29] revised classification, by elevating two of Iljin's [64] subgenera to genus rank, i.e., Babka and Ponticola for the remainder of the 'neogobiin' species. Neilson and Stepien [29] included bathybius in Ponticola in their study without further justification, since they lacked bathybius specimens to sequence and did not hypothesize a nominal genus.
Recent phylogenetic analyses by Zarei et al. [30] and Tajbakhsh et al. [69] provided support for the reclassification of Gobius bathybius from Ponticola to Neogobius sensu stricto [29]. Neogobius bathybius however, differs from the other four Neogobius species in its cheek sensory papillae pattern by possessing one additional transverse row before row b (i.e., five transverse suborbital rows before row b; Fig 11) [68]. The presence of eight transverse suborbital rows and five before row b might be interpreted as a synapomorphy with Mesogobius, but N. bathybius possesses two transverse rows below row b, a plesiomorphic feature shared with Neogobius, Ponticola (except for P. syrman that has three rows), and Proterorhinus; this differs from Mesogobius, which has three rows. The suborbital lateral line system pattern of Mesogobius and N. bathybius do not completely match and might be attributed to parallel evolution or to two step development, where the first step was the synapomorphy of an additional transverse suborbital row in front of suborbital row b, and the second step was the addition of one more transverse suborbital row below row b. In terms of otolith morphology, the otoliths of all Neogobius species except for bathybius are characterized by having a square-rhomboid (N. caspius and N. melanostomus) to a discoid-rhomboid (N. fluviatilis and N. pallasi) shape, a convex and regularly curved dorsal rim, a posterodorsal projection that is usually long and narrow, tapering or rounded, and strongly bent outwards, δ 24.3-33.4˚, α 14.7-22.5˚, and presence of subcaudal iugum and dorsal depression. On the other hand, the N. bathybius otolith is characterized by having a two-humped long rectangle shape, a highly positioned posterior hump, a dorsal rim with a deep broad V-shaped concavity, a posterodorsal projection that is bulky and very broad and does not bend outwards, δ 13.5-22.1˚, α 2.1-10.6˚, and absence of subcaudal iugum and dorsal depression. Among the otoliths of all Ponto-Caspian endemic gobiids studied here, a dorsal rim with two angular humps (with the posterior hump being highly positioned) and a deep broad V-shaped concavity, and a bulky and very broad posterodorsal projection are found in only one other species, Mesogobius nunultimus. Zarei et al. [30] suggest that as an alternative to the mitochondrial phylogenetic hypothesis [30,69], an ancient hybridization scenario between Neogobius melanostomus and Mesogobius nunultimus might have led to the same phylogenetic pattern as the hypothesized sister group relationship between bathybius and N. melanostomus. In addition to the suborbital papillae pattern and the otolith shape, N. bathybius shows several other intermediate morphological and ecological characteristics (Table 13). However, its intermediate characters (best exemplified by the intermediate number of vertebrae, second dorsal and anal fin elements, longitudinal scale rows, head, body and caudal peduncle depths, longevity, and migration) and those completely different from the characters of the putative parental species (e.g., first dorsal elements, growth, habitat, depth, and spawning period; Table 13) could be the result of hybridization and later isolating evolution, as well as the outcome of other scenarios. Therefore, we suggest a chromosome analysis and genomic approach to resolve the generic classification of Neogobini and Ponticolini.

Phylogenetic placement of Anatirostrum and Benthophiloides
The morphologically well-defined benthophilin group is characterized by several apomorphies: (i) suborbital row 5i longer than row 6i, extending below rear termination of row d, with the row 6i separated from row d, (ii) suborbital row 4 not ascending above the level of row b, (iii) interorbital papillae present, (iv) loss of all head canals, (v) a tubular anterior nostril without a rim process, and (vii) absence or, at least, reduction in scale cover, with no squamation on head. Hitherto, the two phylogenetically studied benthophilin genera, Benthophilus and Caspiosoma, formed a monophyletic clade referred to by Neilson and Stepien [29] as Benthophilini. The otoliths of the benthophilin group are characterized by a reduced, shallow sulcus that lacks well-developed ostial lobes and has no subcaudal iugum. Each of the four benthophilin genera possess a distinct otolith shape, reflecting their generic classification and phylogenetic affinities: long elliptical in Benthophilus, right trapezoid in Anatirostrum, long rectangle in Benthophiloides, and pentagonal in Caspiosoma.
Caspiosoma caspium, having the most distinctive otolith shape among the four benthophilin genera, has a basal phylogenetic placement within Benthophilini [29]. Absence of scales or scale-like derivatives in Caspiosoma is a reductive feature shared with Benthophiloides; however, the otoliths of the two genera differ chiefly in the shape of dorsal rim above the cauda (angular vs. convex and gently curved, highest above cauda) and the inclination of ostium (inclined at 7.5˚vs. not inclined). Also, a significant number of morphological features link Benthophilus with Anatirostrum [34], but Anatirostrum lacks the chin barbel and dermal fold at the jaw angle of Benthophilus as well as the large tubercles seen in Benthophilus. Within the benthophilin group, Anatirostrum shows a number of autapomorphies including an additional suborbital row below level of suborbital longitudinal row b, an elongated and duck-bill shaped snout with posterior nostrils displaced well anterior to the orbit, and a complete sequence of pterygiophores between the first and second dorsal fins. Furthermore, the otoliths of Anatirostrum and Benthophilus differ mainly in the dorsal rim's curvature (horizontal to slightly concave vs. convex and gently curving), and the shape of the predorsal angle (orthogonal and slightly raised vs. obtuse and markedly depressed). Molecular data of Benthophiloides and Anatirostrum are still missing, however, these mosaic patterns of morphological features indicate relatively divergent but sister group relationships between Caspiosoma and Benthophibides, and between Benthophilus and Anatirostrum.

Conclusion
The results indicated high taxonomic efficiency of otolith morphology combined with morphometry at different taxonomic levels for the Ponto-Caspian gobiids. Separation of otoliths at different taxonomic levels requires consideration of different morphological characters and otolith variables [46]. This was also the case in our study. In addition, it also appears that these qualitative and quantitative otolith data contain important phylogenetic signals, however, more studies are needed to complete these evaluations and confirm our otolith study findings.
Supporting information S1 Checklist. Ethical, cultural, and scientific considerations specific to inclusivity in global research. (DOCX)